P5387CRISPR/dCas9 Activated Expression of Cardiomyocyte Differentiation Factors in CDCs in Myocardial Infarctions

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
T Sano ◽  
S Ishigami ◽  
S Bandaru ◽  
T Ito ◽  
S Sano
Keyword(s):  
Transcriptional Activation ◽  
Heart Diseases ◽  
Large Population ◽  
Treatment Strategies ◽  
Neonatal Rat ◽  
Cardiomyocyte Differentiation ◽  
Differentiation Potential ◽  
Regulatory Regions ◽  
Specific Differentiation ◽  
Differentiation Factors

Abstract Background Existing therapies against myocardial infarction (MI) involve disease management by preventing additional damage to the heart muscle. However, new treatment strategies are in greater demand, which deems to focus on restoring cardiac function by replacing the damaged cells after MI, rather than merely manage the disease. Cardiosphere-derived cells (CDCs) have emerged as a potential source of cardiac regenerative therapy. In spite of being a promising option, the poor differentiation potential of CDCs to develop into a functional population of cardiomyocytes has always been a significant setback. Purpose The purpose of the present study centers to overcome the aforementioned setback by enhancing the efficiency of rat CDCs to develop into a large population of cardiomyocytes by intrinsic activation of cardio-specific differentiation factors (TNNT2, GATA4, Mef2c) by Crispr/dcas9 assisted transcriptional enhancement system. Methods In the foremost step, an exhaustive screening was performed to identify the specific sequences in endogenous regulatory regions (enhancers and promoters) responsible for transcriptional activation of the TNNT2 gene. Once, potential regulatory regions at proximal and distal end of TNNT2 were identified, crRNAs were designed complementing these regions for recruiting Crispr/dcas9 system fused with transcriptional activator like VP64 (CRISPR-dCas9-VP64). Two distinct plasmids were constructed with crRNA (RFP fused) inserts and CRISPR-dCas9-VP64 (GFP fused) followed by transfection in CDCs those isolated from the heart of a neonatal rat. Post transfection, CDCs were then analyzed for the quantitative expression of cardiomyocyte differentiation factors as well as for fibroblast differentiation factors in comparison with un-transfected CDCs. Results We identified a panel of specific crRNA targeting the enhancers and promoters which demonstrated significantly higher expression of differentiation factors like troponin, GATA4, and Mef2c. Further, the fluorescent visualization with GFP and RFP was prominent in the CDCs confirming that these panel of crRNA enhanced the expression of differentiation factors compared to the un-transfected counterparts. Interestingly, the same panel crRNA, in contrast, demonstrated diminished expression of fibroblast differentiation factors like Col1A1, clearly emphasizing that the CRISPR dCas9 system recruitment at regulatory regions forms an efficient molecular targeting system for enhancing the differentiation potential of CDCs into cardiomyocytes. Conclusion We have identified endogenous regulatory regions responsible for an intrinsic activation of cardio-specific differentiation factors assisted by Crispr/dcas9 gene transcriptional system. We anticipate the method developed herein can enhance and cardiomyogenic efficiency of CDCs to differentiate into a large population of cardiomyocytes to treat Ischemic heart diseases.

Non-invasive ultrasound-based imaging of atherosclerosis

VASA ◽  
2019 ◽  
Vol 48 (2) ◽  
pp. 126-133 ◽  
Author(s):  
Mathias Kaspar ◽  
Iris Baumgartner ◽  
Daniel Staub ◽  
Heinz Drexel ◽  
Christoph Thalhammer
Keyword(s):  
Large Population ◽  
Treatment Strategies ◽  
Intima Media Thickness ◽  
Scientific Work ◽  
Flow Mediated Dilation ◽  
Non Invasive ◽  
Grey Scale ◽  
Current Review ◽  
Appropriate Treatment ◽  
Contrast Enhanced

Abstract. Early detection of vascular damage in atherosclerosis and accurate assessment of cardiovascular risk factors are the basis for appropriate treatment strategies in cardiovascular medicine. The current review focuses on non-invasive ultrasound-based methods for imaging of atherosclerosis. Endothelial dysfunction is an accepted early manifestation of atherosclerosis. The most widely used technique to study endothelial function is non-invasive, flow-mediated dilation of the brachial artery under high-resolution ultrasound imaging. Although an increased intima-media thickness value is associated with future cardiovascular events in several large population studies, systematic use is not recommended in clinical practice for risk assessment of individual persons. Carotid plaque analysis with grey-scale median, 3-D ultrasound or contrast-enhanced ultrasound are promising techniques for further scientific work in prevention and therapy of generalized atherosclerosis.

scholarly journals Cardiooncology—dealing with modern drug treatment, long-term complications, and cancer survivorship

2021 ◽  
Author(s):  
Claudia de Wall ◽  
Johann Bauersachs ◽  
Dominik Berliner
Keyword(s):  
Side Effects ◽  
Checkpoint Inhibitors ◽  
Heart Diseases ◽  
Tumor Therapy ◽  
Treatment Strategies ◽  
Tumor Treatment ◽  
Cardiac Side Effects ◽  
Cardiovascular Side Effects ◽  
Modern Drug

AbstractModern treatment strategies have improved prognosis and survival of patients with malignant diseases. The key components of tumor treatment are conventional chemotherapy, radiotherapy, targeted therapies, and immunotherapy. Cardiovascular side-effects may occur in the early phase of tumor therapy or even decades later. Therefore, knowledge and awareness of acute and long-lasting cardiac side effects of anti-cancer therapies are essential. Cardiotoxicity impairs quality of life and overall survival. The new cardiologic subspecialty ‘cardio-oncology’ deals with the different cardiovascular problems arising from tumor treatment and the relationship between cancer and heart diseases. Early detection and treatment of cardiotoxicity is of crucial importance. A detailed cardiac assessment of patients prior to administration of cardiotoxic agents, during and after treatment should be performed in all patients. The current review focusses on acute and long-term cardiotoxic side effects of classical cytotoxic and selected modern drug treatments such as immune checkpoint inhibitors and discusses strategies for the diagnosis of treatment-related adverse cardiovascular effects in cancer patients.

The Aspergillus nidulans abaA gene encodes a transcriptional activator that acts as a genetic switch to control development

1994 ◽  
Vol 14 (4) ◽  
pp. 2503-2515
Author(s):  
A Andrianopoulos ◽  
W E Timberlake
Keyword(s):  
Aspergillus Nidulans ◽  
Transcriptional Activation ◽  
Expression System ◽  
Regulatory Gene ◽  
Binding Motif ◽  
Mobility Shift ◽  
Genetic Switch ◽  
Regulatory Regions ◽  
Cis Acting ◽  
Gel Mobility Shift

The Aspergillus nidulans abaA gene encodes a protein containing an ATTS DNA-binding motif and is required for the terminal stages of conidiophore development. Results from gel mobility shift and protection, missing-contact, and interference footprint assays showed that AbaA binds to the sequence 5'-CATTCY-3', where Y is a pyrimidine, making both major- and minor-groove contacts. Multiple AbaA binding sites are present in the cis-acting regulatory regions of several developmentally controlled structural genes as well as those of the upstream regulatory gene brlA, the downstream regulatory gene wetA, and abaA itself. These cis-acting regulatory regions confer AbaA-dependent transcriptional activation in a heterologous Saccharomyces cerevisiae gene expression system. From these observations, we propose that the AbaA transcription factor establishes a novel set of feedback regulatory loops responsible for determination of conidiophore development.

A gene therapeutic approach to inhibit calcium and integrin binding protein 1 ameliorates maladaptive remodelling in pressure overload

2018 ◽  
Vol 115 (1) ◽  
pp. 71-82 ◽  
Author(s):  
Andrea Grund ◽  
Malgorzata Szaroszyk ◽  
Janina K Döppner ◽  
Mona Malek Mohammadi ◽  
Badder Kattih ◽  
...  
Keyword(s):  
Heart Failure ◽  
Cardiac Hypertrophy ◽  
Cardiac Function ◽  
Binding Protein ◽  
Treatment Strategies ◽  
Pressure Overload ◽  
Cellular Growth ◽  
Neonatal Rat ◽  
Rat Cardiomyocytes ◽  
Pathological Cardiac Hypertrophy

Abstract Aims Chronic heart failure is becoming increasingly prevalent and is still associated with a high mortality rate. Myocardial hypertrophy and fibrosis drive cardiac remodelling and heart failure, but they are not sufficiently inhibited by current treatment strategies. Furthermore, despite increasing knowledge on cardiomyocyte intracellular signalling proteins inducing pathological hypertrophy, therapeutic approaches to target these molecules are currently unavailable. In this study, we aimed to establish and test a therapeutic tool to counteract the 22 kDa calcium and integrin binding protein (CIB) 1, which we have previously identified as nodal regulator of pathological cardiac hypertrophy and as activator of the maladaptive calcineurin/NFAT axis. Methods and results Among three different sequences, we selected a shRNA construct (shCIB1) to specifically down-regulate CIB1 by 50% upon adenoviral overexpression in neonatal rat cardiomyocytes (NRCM), and upon overexpression by an adeno-associated-virus (AAV) 9 vector in mouse hearts. Overexpression of shCIB1 in NRCM markedly reduced cellular growth, improved contractility of bioartificial cardiac tissue and reduced calcineurin/NFAT activation in response to hypertrophic stimulation. In mice, administration of AAV-shCIB1 strongly ameliorated eccentric cardiac hypertrophy and cardiac dysfunction during 2 weeks of pressure overload by transverse aortic constriction (TAC). Ultrastructural and molecular analyses revealed markedly reduced myocardial fibrosis, inhibition of hypertrophy associated gene expression and calcineurin/NFAT as well as ERK MAP kinase activation after TAC in AAV-shCIB1 vs. AAV-shControl treated mice. During long-term exposure to pressure overload for 10 weeks, AAV-shCIB1 treatment maintained its anti-hypertrophic and anti-fibrotic effects, but cardiac function was no longer improved vs. AAV-shControl treatment, most likely resulting from a reduction in myocardial angiogenesis upon downregulation of CIB1. Conclusions Inhibition of CIB1 by a shRNA-mediated gene therapy potently inhibits pathological cardiac hypertrophy and fibrosis during pressure overload. While cardiac function is initially improved by shCIB1, this cannot be kept up during persisting overload.

Abstract 3857: Structural Heterogeneities are a Substrate for Re-excitation, Reflection, and Arrhythmogenesis in Cardiac Muscle

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
David S Auerbach ◽  
Sergey Mironov ◽  
Jose Jalife
Keyword(s):  
Cardiac Muscle ◽  
Ventricular Myocytes ◽  
Heart Diseases ◽  
Distal Region ◽  
Neonatal Rat ◽  
Excitable Tissue ◽  
Source To Sink ◽  
Replacement Fibrosis ◽  
Electrical Impulses ◽  
Neonatal Rat Ventricular Myocytes

Background: Heart diseases predispose to arrhythmias and sudden cardiac death by mechanisms that are poorly understood. We tested the hypothesis that in fully excitable tissue abrupt geometrical expansions resulting from varying wall thickness, replacement fibrosis, ischemia, or accessory pathways, set the stage for to-and-fro propagation (reflection) of electrical impulses over the same pathway, leading to premature excitation and reentry initiation. Methods: We used patterned monolayers of cultured neonatal rat ventricular myocytes consisting of two wide regions connected by a fully excitable, thin isthmus (0.1, 0.5, 1, & 2 mm wide, 6 mm long). We compared control monolayers with those overexpressing Na channels (NaCh, Ad-hSCN5a). Impulse propagation was optically imaged (Di-8-ANEPPS) at high resolution. Results: Impulses initiated proximally in a wide region propagated 1:1 through relatively broad isthmuses (2 mm) and then into the distal expansion at higher frequencies than through narrower isthmuses (0.1 mm). In control monolayers, with relatively low excitability, the prevalence of reflection was small (15%, n=61). NaCh overexpression increased excitability. It also increased the incidence of reflection (38%, n=26). In homogeneous monolayers, NaCh overexpression increased the conduction velocity (15–17%) and prolonged the action potential duration (APD, 21–26%) at the frequencies (2– 4 Hz) at which reflection occurred. During reflection, the APD at the distal expansion was prolonged, compared to APDs within the isthmus or distally when there was no reflection. APD prolongation provided a substrate for local phase-3 re-excitation at the isthmus, and thus reflection. In some cases, reflection was sustained over several consecutive beats. Reflection also triggered the initiation of reentry; as the beat reflected back, a small distal region was re-excited, resulting in unidirectional propagation and the initiation of reentry. Reflection was never observed in structurally homogeneous monolayers, whether with or without the presence of NaCh overexpression. Conclusion: Source-to-sink mismatch in areas of cardiac muscle expansion creates APD heterogeneities, which may serve as a substrate for reflection and arrhythmogenesis.

Abstract 1457: IGFBP-4-Mediated Wnt Inhibition is Required for Cardiac Myogenesis

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Weidong Zhu ◽  
Ichiro Shiojima ◽  
Li Zhi ◽  
Hiroyuki Ikeda ◽  
Masashi Yoshida ◽  
...  
Keyword(s):  
Growth Factor ◽  
Wnt Signaling ◽  
Heart Diseases ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cardiomyocyte Differentiation ◽  
Canonical Wnt Signaling ◽  
Canonical Wnt

Insulin-like growth factor-binding proteins (IGFBPs) bind to and modulate the actions of insulin-like growth factors (IGFs). Although some of the effects of IGFBPs appear to be independent of IGFs, the precise mechanisms of IGF-independent actions of IGFBPs are largely unknown. In this study we demonstrate that IGFBP-4 is a novel cardiogenic growth factor. IGFBP-4 enhanced cardiomyocyte differentiation of P19CL6 embryonal carcinoma cells and embryonic stem (ES) cells in vitro. Conversely, siRNA-mediated knockdown of IGFBP-4 in P19CL6 cells or ES cells attenuated cardiomyocyte differentiation, and morpholino-mediated knockdown of IGFBP-4 in Xenopus embryos resulted in severe cardiac defects and complete absence of the heart in extreme cases. We also demonstrate that the cardiogenic effect of IGFBP-4 was independent of its IGF-binding activity but was mediated by the inhibitory effect on canonical Wnt signaling. IGFBP-4 physically interacted with a Wnt receptor Frizzled 8 (Frz8) and a Wnt co-receptor low-density lipoprotein receptor-related protein 6 (LRP6), and inhibited the binding of Wnt3A to Frz8 and LRP6. Moreover, the cardiogenic defects induced by IGFBP-4 knockdown both in vitro and in vivo was rescued by simultaneous inhibition of canonical Wnt signaling. Thus, IGFBP-4 is an inhibitor of the canonical Wnt signaling, and Wnt inhibition by IGFBP-4 is required for cardiogenesis. The present study provides a molecular link between IGF signaling and Wnt signaling, and suggests that IGFBP-4 may be a novel therapeutic target for heart diseases.

Abstract 340: From Stem Cells to Cardiomyocytes: HDAC1 Induces Cardiovascular Differentiation by Regulating SOX17-BMP2 Expression in Early Development.

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Eneda Hoxha ◽  
Erin Lambers ◽  
Veronica Ramirez ◽  
Prasanna Krishnamurthy ◽  
Suresh Verma ◽  
...  
Keyword(s):  
Stem Cells ◽  
Molecular Mechanisms ◽  
Es Cells ◽  
Ips Cells ◽  
Full Potential ◽  
Pluripotent Cells ◽  
Differentiation Potential ◽  
Histone Deacetylase 1 ◽  
Specific Differentiation ◽  
Induced Pluripotent

Cardiomyocytes derived from embryonic and induced pluripotent stem cells (ES/iPS) provide an excellent source for cell replacement therapies following myocardial ischemia. However, some of the obstacles in the realization of the full potential of iPS/ES cells arise from incomplete and poorly understood molecular mechanisms and epigenetic modifications that govern their cardiovascular specific differentiation. We identified Histone Deacetylase 1 (HDAC1) as a crucial regulator in early differentiation of mES and iPS cells. We propose a novel pathway in which HDAC1 regulates cardiovascular differentiation by regulating SOX17 which in turn regulates BMP2 signaling in differentiating pluripotent cells. Utilizing stable HDAC1 knock-down (HDAC1-KD) cell lines, we report an essential role for HDAC1 in deacetylating regulatory regions of pluripotency-associated genes during early cardiovascular differentiation. HDAC1-KD cells show severely repressed cardiomyocyte differentiation potential. We propose a novel HDAC1-BMP2-SOX17 dependent pathway through which deacetylation of pluripotency associated genes leads to their suppression and allows for early cardiovascular-associated genes to be expressed and differentiation to occur. Furthermore, we show that HDAC1 affects DNA methylation both during pluripotency and differentiation and plays a crucial, non-redundant role in cardiovascular specific differentiation and cardiomyocyte maturation. Our data elucidates important differences between ES and iPS HDAC1-KD cells that affect their ability to differentiate into cardiovascular lineages. As varying levels of chromatin modifying enzymes are likely to exist in patient derived iPS cells, understanding the molecular circuitry of these enzymes in ES and iPS cells is critical for their potential therapeutic applications in regenerative medicine. Further research in the molecular mechanisms involved in this process will greatly aid our understanding of the epigenetic circuitry of pluripotency and differentiation in pluripotent cells.

Proliferation and differentiation potential of rat forebrain oligodendroglial progenitors both in vitro and in vivo

Development ◽  
1991 ◽  
Vol 111 (4) ◽  
pp. 1061-1080 ◽  
Author(s):  
R. Hardy ◽  
R. Reynolds
Keyword(s):  
Progenitor Cell ◽  
Neonatal Rat ◽  
Myelin Proteins ◽  
Brdu Incorporation ◽  
Cell Type ◽  
Differentiation Potential ◽  
Serum Free ◽  
Rat Forebrain

We have followed the development of the O-2A progenitor cell from the neonatal rat forebrain, both in dissociated cell culture and in cryostat sections, using immunocytochemical techniques employing a panel of antibodies that recognise the cells at different stages of their development. This included the monoclonal antibody LB1, which binds to the surface ganglioside GD3 expressed on O-2A progenitor cells. In secondary cultures enriched for O-2A progenitors maintained in a serum-free chemically defined medium, a large proportion of the cells are primed to differentiate into oligodendroglia and go on to express the oligodendroglial specific surface glycolipid galactocerebroside (GC) and then the myelin proteins CNP and MBP. However, a significant proportion of immature bipolar GD3+ cells remained after 6 days in secondary culture. It appears that not all the O-2A progenitors in our cultures differentiate immediately and some cells remain in an undifferentiated state and divide to replenish progenitor numbers. We have also identified in our cultures a small apolar GD3- cell, which when isolated differentiated into a GD3+ bipolar O-2A progenitor cell. We have termed this cell type a preprogenitor. The differentiation of this cell type into O-2A progenitors may be the source of the immature GD3+ cells present at the later stages of our secondary cultures. The proliferative profile of the cultures was studied using 5′bromo-2-deoxyuridine (BrdU) incorporation as an index of mitosis. Only the immature, bipolar O-2A progenitors were seen to divide at any time in serum-free culture. Neither the more mature multipolar O-2A cells nor the oligodendroglia were seen to divide. The developmental profile of the O-2A cells in the rat forebrain in vivo showed a largely similar progression to that in culture, with a time lag of at least 6 days between GD3 expression and the onset of myelination. BrdU incorporation studies in vivo also showed that the GD3+ progenitor cell is mitotic whereas the GC(+)-expressing oligodendroglia is not. We have shown that there are several significant alterations in the timing of antigen expression in both O-2A progenitors and oligodendroglia in vitro compared to that seen in vivo.

scholarly journals MLL1 is required for PAX7 expression and satellite cell self-renewal in mice

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Gregory C. Addicks ◽  
Caroline E. Brun ◽  
Marie-Claude Sincennes ◽  
John Saber ◽  
Christopher J. Porter ◽  
...  
Keyword(s):  
Satellite Cell ◽  
Satellite Cells ◽  
Transcriptional Activation ◽  
Cell Activation ◽  
Target Genes ◽  
Cell Function ◽  
Skeletal Muscle Regeneration ◽  
Differentiation Potential ◽  
Self Renewal

Abstract PAX7 is a paired-homeobox transcription factor that specifies the myogenic identity of muscle stem cells and acts as a nodal factor by stimulating proliferation while inhibiting differentiation. We previously found that PAX7 recruits the H3K4 methyltransferases MLL1/2 to epigenetically activate target genes. Here we report that in the absence of Mll1, myoblasts exhibit reduced H3K4me3 at both Pax7 and Myf5 promoters and reduced Pax7 and Myf5 expression. Mll1-deficient myoblasts fail to proliferate but retain their differentiation potential, while deletion of Mll2 had no discernable effect. Re-expression of PAX7 in committed Mll1 cKO myoblasts restored H3K4me3 enrichment at the Myf5 promoter and Myf5 expression. Deletion of Mll1 in satellite cells reduced satellite cell proliferation and self-renewal, and significantly impaired skeletal muscle regeneration. Pax7 expression was unaffected in quiescent satellite cells but was markedly downregulated following satellite cell activation. Therefore, MLL1 is required for PAX7 expression and satellite cell function in vivo. Furthermore, PAX7, but not MLL1, is required for Myf5 transcriptional activation in committed myoblasts.

scholarly journals Epidemiology of Heart Disease of Uncertain Etiology: A Population Study and Review of the Problem

Medicina ◽  
2019 ◽  
Vol 55 (10) ◽  
pp. 687 ◽  
Author(s):  
Alessandro Menotti ◽  
Paolo Emilio Puddu
Keyword(s):  
Risk Factors ◽  
Heart Disease ◽  
Population Study ◽  
Serum Cholesterol ◽  
Heart Diseases ◽  
Large Population ◽  
Hdl Cholesterol ◽  
Cox Proportional Hazards ◽  
Italian Population ◽  
Uncertain Etiology

Background and objectives: Previous epidemiological studies have identified a group of heart diseases (here called heart diseases of uncertain etiology—HDUE) whose characteristics were rather different from cases classified as coronary heart disease (CHD), but frequently confused with them. This analysis had the purpose of adding further evidence on this issue based on a large population study. Materials and Methods: Forty-five Italian population samples for a total of 25,272 men and 21,895 women, free from cardiovascular diseases, were examined with measurement of some risk factors. During follow-up, CHD deaths were those manifested as myocardial infarction, other acute ischemic attacks, and sudden death of probable coronary origin, after reasonable exclusion of other causes. Cases of HDUE were those manifested only as heart failure, chronic arrhythmia, and blocks in the absence of typical coronary syndromes. Cox proportional hazards models were computed separately for CHD and HDUE, with 11 risk factors as possible predictors. Results: During an average of 7.4 years (extremes 1–16) there were 223 CHD and 150 HDUE fatal events. Male sex, age, smoking habits, systolic blood pressure, serum cholesterol, and plasma glucose were significantly and directly related to CHD events, while high density lipoprotein (HDL) cholesterol was so in an inverse way. The same risk factors were predictive of HDUE events except serum cholesterol and HDL cholesterol. Multivariable hazards ratio of serum cholesterol (delta = 1 mmol/L) was higher in the CHD model (1.24, 95% CI 1.11–1.39) than in the HDUE model (1.03, 0.5% C.I. 0.89–1.19) and the difference between the respective coefficients was statistically significant (p = 0.0444). Age at death was not different between the two end-points. Conclusions: CHD and HDUE are probably two different morbid conditions, only the first one is likely bound to gross atherosclerotic lesions of coronary arteries and linked to blood lipid levels. We reviewed the problem in epidemiological investigations and addressed inflammation as a potential cofactor to differentiate between CHD and HDUE.

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